Composition for regenerative treatment of cartilage disease

ABSTRACT

A composition for regenerative treatment of cartilage disease, which comprises a PDE4 inhibitor as an active ingredient, specifically a composition comprising a PDE4 inhibitor and a biocompatible and biodegradable polymer is provided, which composition, when formulated into a form suited to administer locally to affected cartilage region, such as microsphere preparation, can provide a pharmaceutical composition showing an excellent effect in regenerative treatment of cartilage.

This is a division of application Ser. No. 10/478,432, filed Nov. 21,2003 now abandoned, which is a §371 of PCT/JP02/04930, filed May 22,2002, and claims the benefit of priority of 2001-154048 filed May 23,2001 in Japan, all of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a composition for regenerativetreatment of cartilage disease, specifically, to a pharmaceuticalcomposition for regenerative treatment of cartilage disease such asosteoarthrosis (degenerative joint disease), chondrodystrophy,degenerative discopathy or meniscus injury.

BACKGROUND ART

Cartilage is considerably elastic that plays a role in the constructionof skeleton together with bone and the protection of internal organs.Cartilage tissue consists of chondrocytes and cartilage matrixsurrounding the same.

Cartilage is formed by mesenchyme-originated chondroblasts which cellsproduce matrix in circumference in the process of cell division andgrowth. The cartilage matrix consists of amorphous matrix and fibrouscomponents, and is classified into the following groups according to theratio of components: (1) hyaline cartilage (articular cartilage,costicartilage, thyroid cartilage etc.);(2) fibrocartilage (discusintervertebrali, pubic symphysis etc.); and (3) elastic cartilage(pharynx lid cartilage, cartilage of acoustic meatus, auricularcartilage, etc.) IGAKU-DAIJITEN, 18th edition, published by Nanzando,pp. 1542.

The main components of cartilage matrix are proteoglycan and collagen(Type II, Type IX and the like). It is known that proteoglycanparticipates in the imbibition (swelling) nature peculiar to cartilagetissue, and collagen in the rigidity of cartilage against the tensionand shearing force.

In proteoglycan of cartilage matrix, it is considered thatglucosaminoglycans such as chondroitin sulfate, keratan sulfate areconnected with a core protein of about 220,000 molecular weight to formmacromolecules, wherein glucosaminoglycans hydrates many watermolecules, which contributes to the imbibition nature of cartilage. TheBone, Vol. 4, pp. 8 (1994).

Articular cartilage has a calcification layer at the transmigrationregion with bone tissue, and, after the completion of growth, nutrientsare supplied to chondrocytes from synovial fluid and are hardly supplieddirectly from blood. In addition, articular cartilage is formed fromhyaline cartilage of high cell differentiation degree, and hence is asensitive organ with extremely low regenerative ability.

The surface of articular cartilage is covered by highly viscous synovialfluid, and by virtue of lubrication mechanism of lubricant comprising asa principal component hyaluronic acid-protein complex, the smooth jointkinematics is maintained. However, it is considered that there isso-called durability in, articular cartilage, and alteration of jointswith aging is unavoidable physiological phenomenon.

Examples of known diseases caused by cartilage disorder includeosteoarthrosis, chondrodystrophy, degenerative discopathy or meniscusinjury.

Among them, osteoarthrosis is a disease wherein a proliferative changeof bone and articular cartilage occurs on the basis of a regressivechange of tissue constituting a joint, mainly, articular cartilage,finally leading to a remarkable morphological change of the joint, whichdisease has markedly increased with the aging of population. Inparticular, the knee joint anthropathy can prevent patients frommaintaining the standing position or walking normally as the pathologyprogresses, and lead to the significant decrease of their ADL (Abilityof Daily Life) which possibly results in a bedridden condition.

Treatment of osteoarthrosis can be classified mainly into conservativetreatment and surgical therapy. Conservative treatment is carried out bythe following methods, for example, (1) administration of non-steroidalantiinflammatory analgesic; (2) thermotherapy; (3) control of weight;(4) therapy with braces; (5) intra-articular infusion of steroidalantiinflammatory analgesic; (6) intra-articular infusion of hyaluronateformulation. In cases wherein conservative treatment is ineffective, orthe disease is in progressed or terminal stage, surgical therapy isconducted by (a) arthroscopic irrigation surgery; (b) high tibialosteotomy or (c) artificial joint replacement, and the like. Senilityand Disorder, Vol. 10, 2nd. issue, pp. 61-69, (1997) & 6th issue, pp.66-77 (1997).

There are various compounds having PDE4 inhibitory activity, which cansuppress the release of inflammatory mediator by inhibiting the PDE4activity. J. Mol. Cell. Cardiol., 12 (Suppl.II), S61 (1989).

It is described that a compound having PDE4 inhibitory activitysuppresses the production of TNF-α which is a cytokine released frommononuclear phagocytes in response to immunostimulants, and is useful intreatment of various inflammatory diseases caused by TNF-α. JP2000-503678A, JP 2000-502724A, JP 2000-510105A, JP 2000-514804A,2000-502350A, JP 2000-501741A, and the like.

However, it has not been known that PDE4 inhibitor is effective forreparative treatment of cartilage diseases at all.

DISCLOSURE OF INVENTION

As stated above, cartilage is known to have extremely low regenerativeability, and it was considered that, once damaged, the regenerationthereof is almost impossible. The conventional pharmacotherapy was onlyconservative treatment which restrains the progressing of disorder.Accordingly, it has long been demanded the development ofpharmacotherapy and/or pharmaceutical agent that enables to conductregenerative treatment of cartilage diseases.

The present inventors have first found that PDE4 is produced bychondrocytes and then compounds having PDE4 inhibitory activity showactivity on cartilage diseases. The inventors have intensively studiedand found that the said PDE4 inhibitors are useful in regenerativetreatment of cartilage diseases, and established the present invention.

The present invention provides a composition for regenerative treatmentof cartilage disease, which comprises a PDE4 inhibitor as an activeingredient. In particular, the present invention provides apharmaceutical preparation suited to administer locally to the site ofcartilage disease, specifically, a composition for regenerativetreatment of cartilage disease in the form of microsphere preparation.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the cAMP hydrolyzing activity in each fractionobtained by fractionating rabbit articular chondrocyte extract by Mono QSepharose column chromatography, in the presence of Compound (44) (∘)and absence of Compound (44) (●).

FIG. 2 is a copy of microphotograph showing the results of observationunder microscope of regeneration of old rabbit articular cartilage inthe presence of microsphere containing Compound (1) or free of Compound(1).

FIG. 3 is a graph showing the cAMP or DGMP hydrolyzing activity in eachfraction obtained by fractionating human articular chondrocyte extractby Mono Q Sepharose column chromatography.

FIG. 4 is a graph showing the inhibitory activity (IC₅₀) of PDE4inhibitor toward fractions 28-30 that showed potent cAMP hydrolyzingactivity as demonstrated in FIG. 3.

FIG. 5 is a graph showing the in vitro drug elution characteristics ofmicrospheres obtained in Examples 1, 2 and 3.

FIG. 6 is a graph showing the time-course of plasma concentration ofCompound (1) administered to a rat intravenously. Data are shown by mean± standard deviation (n=3).

FIG. 7 is a graph showing the time-course of plasma concentration of anactive ingredient following the subcutaneous injection of microspheredispersion obtained in Example 1-(5), 2-(2) or 3-(2) into a rat. Dataare shown by mean±standard deviation (n=5).

FIG. 8 is a graph showing the time-course of Compound (1) remaining inthe preparation following the subcutaneous injection of microspheredispersion obtained in Example 2-(2) into a rat. Data are shown bymean±standard deviation (n=5).

FIG. 9 is a graph showing the time-course of Compound (2) remaining inthe preparation following the subcutaneous injection of microspheredispersion obtained in Example 6-(5) or 7-(2) into a rat. Data are shownby mean± standard deviation (n=4).

BEST MODE FOR CARRYING OUT THE INVENTION

The composition of the present invention for regenerative treatment ofcartilage disease can enhance the expression of cartilage matrix proteinencoding gene and thereby showing superior matrix production promotingeffect on cartilage especially on articular cartilage that has extremelylow regenerative activity, and cure cartilage diseases through theregeneration of cartilage.

As herein used, the term “regenerative treatment of cartilage disease”refers to treatment not only for arresting the progress of cartilagedisease but also for restoring a cartilage undergone deformation and/ordetrition due to illness, lesion, or the like to the original state.

The pharmaceutical composition of the present invention can be preparedby combining a PDE4 inhibitor as an active ingredient and a conventionalpharmaceutically acceptable excipient or a diluting agent therefor.Preferred pharmaceutical composition is a sustained release compositionfor local administration, which contains a PDE4 inhibitor(s) and abiocompatible and biodegradable polymer(s). The composition for localadministration is preferably in the form of depot formulation, and morepreferably in the form of microsphere, which microsphere can beformulated as an injectable preparation.

Examples of PDE4 inhibitor usable as an active ingredient ofpharmaceutical compositions of the present invention include all thecompounds having PDE4 inhibitory activity, for example, those describedin JP 05-229987A (1993), JP 09-59255A (1997), JP 10-226685A (1998), EP158380, WO/94/25437, U.S. Pat. No. 5,223,504, WO/95/4045, EP 497564, EP569414, EP 623607, EP 163965, U.S. Pat. No. 5,605,914, WO/95/35282,WO/96/215, U.S. Pat. Nos. 5,804,588,5,552,438, WO/93/9118, WO/96/31485,EP 459505, WO/97/22585, EP 738715, WO/91/16314, WO/96/218, WO/97/18208,EP 158380, WO/99/50270, EP 260817, WO/98/11113, WO/94/22852, EP 432856,U.S. Pat. No. 4,193,926, WO/98/13348, WO/96/6843, JP 2000-503678A(WO/98/14432), JP 2000-502724A (WO/98/9961), JP 2000-510105A(WO/97/40032), JP 2000-514804A (WO/98/2440), JP 2000-502350A(WO/97/23457), JP 2000-501741A (WO/97/2585), and the like.

PDE can be classified into PDE1-5 according to the teaching of “Trendsin Pharmacological Sciences, vol. 11, pp. 150-155”, and PDE4 inhibitorssuitable for the present composition for regenerative treatment ofcartilage disease are preferably selective to PDE4 with higherinhibitory activity against PDE4 compared to others (PDE1-3, 5), morepreferably have 10 times or more inhibitory activity on PDE4 than on theother PDEs. The inhibitory activity of such PDE4 inhibitor on PDE4 isparticularly preferably 50 times or more, and yet more preferably 100times or more of that on the other PDEs.

Preferable PDE4 inhibitors are compounds of which IC₅₀ of PDE4inhibitory activity is 0.1-1000 nM, preferably 0.1-100 nM, morepreferably less than 100 nM, when determined by a method described in“Advances in Cyclic Nucleotide Research”, vol. 10, pp. 69-92, 1979,Raven Press.

Specific examples of selective PDE4 inhibitors include Compounds (1) to(57) represented by the following formulas or pharmaceuticallyacceptable salts thereof.

The compounds having PDE4 inhibitory activity can be classified into (A)to (D) below according to the chemical 5 structure, and a PDE4 inhibitorfor the present invention can be selected from these compoundsappropriately; however, preferred compounds belong to (A) and (B), inparticular, (A).

-   (A) Compounds having naphthalene skeleton or a partial structure    analogous thereto [e.g., Compounds (1), (2), (38), (47), and (52) to    (57)];-   (B) Compounds having 3-cyclopentyloxy-4-methoxyphenyl structure or a    partial structure analogous thereto [e.g., Compounds (6), (9), (11),    (12), (14), (17), (19), (20), (21), (24), (25), (26), (27), (33),    (34), (35), (39), (40), (44), (49), (50) and (51)];-   (C) Compounds having a xanthine skeleton or a partial structure    analogous thereto [e.g., Compounds (5), (7), (28), (29), (30), (31),    (32), (36), (37), (41), (43) and (46)]and-   (D) Compounds having a different structure from those described    in (A) to (C) above [e.g., Compounds (3), (4), (8), (10), (13),    (15), (16), (18), (22), (23), (42), (45) and (48)].

Examples of compounds of group (A) include those shown by the followingformulas (I) to (III) and pharmacologically acceptable salts thereof.

Wherein R¹ and R² are the same or different and each a hydrogen atom, ahydroxyl group, a cyclo-lower alkyloxy group, or an optionallysubstituted lower alkoxy group, or bind together at the ends to form alower alkylenedioxy group;

-   R³ is an optionally substituted 6-membered nitrogen-containing    heterocyclic group; and-   —OR⁴ and —OR⁵ are the same or different and each an optionally    protected hydroxyl group. JP 05-229987A, (1993).

Wherein R¹and R² are the same or different and each a hydrogen atom oran optionally protected hydroxyl group; either of R^(3′) and R^(4′) isan optionally protected hydroxy-substituted methyl group and the otheris a hydrogen atom, a lower alkyl group or an optionally protectedhydroxy-substituted methyl group; and

-   R^(5′) and R^(6′) are the same or different and each a hydrogen    atom, an optionally substituted lower alkyl group, an optionally    substituted phenyl group or an optionally protected amino group, or    bind together at the ends and form in association with the adjacent    nitrogen atom an optionally substituted heterocyclic group.    JP-09-59255A, (1993).

Wherein A is a group selected from those shown by the formulas:

wherein R^(1′) and R^(2′) are the same or different and each a hydrogenatom or an optionally protected hydroxyl group; R³¹ is an optionallyprotected hydroxymethyl group; R³² is a hydrogen atom, a lower alkylgroup or an optionally protected hydroxymethyl group; R³³ is anoptionally substituted lower alkyl group; R⁴¹ is an optionally protectedhydroxymethyl group; R⁴² is an optionally protected hydroxymethyl group;the dotted line represents the presence or absence of a double bond; and

-   R^(5′) sand R^(6′) are the same or different and each a hydrogen    atom or an optionally protected amino group, or bind together at the    ends and form in association with the adjacent nitrogen atom an    optionally substituted heterocyclic group. JP-10-226685A (1998).

As a PDE4 inhibitor which is an active ingredient of the presentcomposition for regenerative treatment of cartilage disease, among group(A), compounds having naphthalene or isoquinoline skeleton andpharmaceutically acceptable salts thereof are more preferred, andCompounds (1) and (2) and their pharmaceutically acceptable salts arestill more preferred.

Since PDE4 inhibitors may cause vomiting or gastric acid secretiondepending on dosage when acted systemically (Cellular Signaling, 9(3-4), pp. 227-236 (1997)), the present composition for regenerativetreatment of cartilage disease is preferably applied locally to avicinity of affected region (especially, vicinity of articularcartilage), so that the drug concentration in the systemic blood doesnot increase but the one at the affected cartilage region is maintained.To establish this purpose, it is preferred to formulate the compositioninto a sustained release form which can advantageously reduce thefrequency of administration and also decrease the burden of patients.

Examples of preferred embodiments of the present composition includedepot preparations which gradually release a drug when administeredlocally (e.g., pellet preparation, gel preparation, matrix preparation,microsphere preparation, a sustained release preparation obtained byadding a drug into an aqueous solution of a biocompatible andbiodegradable polymer, a preparation which is designed to be a liquid atthe time of administration and to form a gel in a living body afteradministration, a preparation embedded in various bases which arereported to be generally used in the field of orthopedics, and thelike.)

Examples of pellet preparations include a long-term sustained releasepreparation obtainable by compressing a drug and fine particles oflactic acid-glycolic acid copolymer of which terminal carboxyl group isesterified by an alcohol, and the like. (JP2001-187749A)

Examples of gel preparations include those obtained by dissolving into aphosphate buffer a drug and hyaluronic acid which is chemically bound topolyethylene glycol (Journal of Controlled Release, 59 (1999) pp.77-86), and the like.

Examples of matrix preparations comprising a drug include those obtainedby impregnating a drug into granular material of collagen or fibrousmembrane preparation, or by adding a drug to a granular material ofcollagen or a reaction mixture for preparing a fibrous membranepreparation, and the like (JP10-182499A (1998), JPO6-305983 (1994)).

Examples of a sustained release preparation obtained by adding a druginto an aqueous solution of a biocompatible and biodegradable polymerinclude those obtained by adding a drug into an aqueous sodiumhyaluronate solution, and the like.

Examples of a preparation designed to be a liquid at the time ofadministration and to form a gel in a living body after administrationinclude those wherein a drug and a lactic acid-glycolic acid copolymerare dissolved in N-methyl-2-pyrrolidone (Journal of Controlled Release,33 (1995) pp. 237-243), or a preparation comprising a drug and a polymerthat exists as an solution at low temperature but forms a gel at bodytemperature, such as a block co-polymer of lactic acid-glycolic acidcopolymer and polyethylene glycol and the like (ibid., 27(1993),139-147).

Examples of a preparation embedded in various bases which are reportedto be generally used in the field of orthopaedics include those preparedby mixing a drug and a base (e.g., water-insoluble biocompatible andbiodegradable polymer, polymethyl methacrylate, hydroxyapatite,tricalcium phosphate or the like). Biomaterials, vol. 21, pp. 2405-2412(2000); and International Journal of Pharmaceutics, vol. 206, pp. 1-12(2000).

Preparations for local administration that release an effective amountof PDE4 inhibitor gradually to a vicinity of cartilage region with alesion(s) (especially, vicinity of articular cartilage) are preferred inthe respect that the administration frequency during the term requiredfor regenerative treatment of cartilage disease can be reduced.

Among depot preparations, in the case of microspheres feasible for localadministration by injection, the particle size of such microspheres ispreferably in the range suitable for passing a needle, more preferably0.01-150 μm, particularly preferably 0.1-100 μm in the respect that theirritation at the affection site can be reduced.

Since the present composition for regenerative treatment of cartilagedisease, which comprises a PDE4 inhibitor as an active ingredient, isadministered locally to a vicinity of cartilage region with a lesion(s)(especially, vicinity of articular cartilage), it would be preferable tomake the dosage small. Accordingly, the PDE4 inhibitor content in thecomposition such as microsphere preparation can be preferably 0.0001-80%by weight, more preferably 0.001-50% by weight, and further morepreferably 0.01-50% by weight. The dose of a PDE4 inhibitor as an activeingredient may vary depending on the kind of PDE4 inhibitor to be used,the weight, age, conditions of the subject or a site to be applied andis generally determined by a physician; however, for localadministration, the dose can usually be in the range of from 1 ng to 1 gper affected region.

The composition for regenerative treatment of cartilage disease of thepresent invention can be prepared in a conventional manner using a PDE4inhibitor and a pharmaceutically acceptable excipient or a carriertherefor. Preferred composition can be prepared-by combining a PDE4inhibitor and a biocompatible and biodegradable polymer.

Among them, the water-insoluble biocompatible and biodegradable polymeris a water-insoluble biocompatible and biodegradable polymer thatrequires at least 1000 ml of water to dissolve 1 g of the polymer at 25°C., and specific example include hydroxy fatty acid polyesters andderivatives thereof (for example, poly lactic acid, poly glycolic acid,poly citric acid, poly malic acid, poly-β-hydroxybutyric acid,ring-opening polymerized ε-caprolactones, lactic acid-glycolic acidcopolymer, 2-hydroxybutyric acid-glycolic acid copolymer, blockcopolymer of poly lactic acid and polyethylene glycol, block copolymerof poly glycolic acid and polyethylene glycol, and block copolymer oflactic acid-glycolic acid copolymer and polyethylene glycol, etc.),polymers of alkyl α-cyanoacrylates (e.g., polybutyl-2-cyanoacrylate,etc.), polyalkylene oxalate (e.g., polytrimethylene oxalate,polytetramethylene oxalate, etc.), polyortho-esters, polycarbonates(e.g., polyethylene carbonate, polyethylenepropylene carbonate, etc.),polyortho-carbonates, polyamino acids (e.g., poly-γ-L-alanine,poly-γ-benzyl-L-glutamic acid, poly-γ-methyl-L-glutamic acid, etc.),hyaluronic acid esters. One or more of these polymers can be used. Otherbiocompatible and biodegradable polymers include sodium hyaluronate,chondroitin sulfate, collagen, gelatin, fibrin, and the like.

Among the water insoluble biocompatible and biodegradable polymersabove, hydroxy fatty acid polyesters are particularly preferred. Aboveall, those of which average molecular weight ranging in between 2000 andabout 800000 are more preferred, those ranging in between 2000 and about200000 are especially preferred and those ranging in between 5000 and50000 are most preferred.

In addition, among the hydroxy fatty acid polyesters above, poly lacticacid, lactic acid-glycolic acid copolymer and 2-hydroxybutyricacid-glycolic acid copolymer are more preferred. The molar ratio oflactic acid and glycolic acid in a lactic acid-glycolic acid copolymeris preferably 90:10 to 30:70, more preferably 80:20 to 40:60, and themolar ratio of 2-hydroxybutyric acid and glycolic acid in a2-hydroxybutyric acid-glycolic acid copolymer is preferably 90:10 to30:70, more preferably 80:20 to 40:60.

When formulating a PDE4 inhibitor above into a depot preparation, it canbe carried out appropriately depending on the intended embodiment,optionally after pulverizing a PDE4 inhibitor if necessary.

Pulverization of PDE4 inhibitor can be carried out using any one ofconventional methods for producing fine particles including mechanicalpulverization methods such as jet mill, hammer mill, convolution ballmill, jar ball. mill, beads mill, shaker mill, rod mill and tube millpulverizations, or so-called crystallization method wherein a drug isfirst dissolved in a solvent and then recrystallized by adjusting pH,changing temperature, or altering the constitution of solvent, andrecovering the particles by centrifugation, filtration, or the like.

When preparing the above-mentioned various types of formulations of thepresent pharmaceutical composition, any appropriate process can be useddepending on the selected PDE4 inhibitor.

For example, microsphere preparation can be prepared by the followingmethods. In case that a salt of a PDE4 inhibitor shows low incorporationrate into a microsphere, it may be converted into corresponding freeform using an acid or a base prior to the preparation of microspheres.

(1) In-Water Drying Method

In this method, a drug is added to a solution of water-insolublebiocompatible and biodegradable polymer in a water-immiscible organicsolvent of which boiling point is lower than water (water-insolublepolymer solution), and the resultant organic phase is dispersed into anaqueous phase to give an O/W emulsion, which is followed by removal ofthe organic solvent. This method can be conducted in a manner similar tothose described in, for example, JP 56-19324B (1981), JP 63-91325A(1988), JP 08-151321A (1996),

Kajeev Jain et al., “Controlled Drug Delivery by Biodegradable Poly(Ester) Devices: Different Preparative Approaches”, Drug Development andIndustrial Pharmacy, vol. 24(8), pp. 703-727, 1998, JP 60-100516A(1985), JP 62-201816A (1987), JP 09-221417A (1997) and JP 06-211648A(1994).

(2) Phase Separation Method

In this method, into a solution of water-insoluble biocompatible andbiodegradable polymer in an organic solvent is dissolved or dispersed adrug, or is dispersed an aqueous solution of the drug. A hardening agentis then added gradually with stirring to obtain solid precipitations.This method can be conducted in a manner similar to those described in,for example, JP 60-67417A (1985), U.S. Pat. Nos. 5,503,851, 5,000,886,Eur. J. Pharm. Biopharm. vol. 42 (1), pp. 16-24 (1996) and the forecitedJain et al. 20. (ibid.)

(3) Spray Drying Method

In this method, to a solution of water insoluble biocompatible andbiodegradable polymer in an organic solvent is dissolved or dispersed adrug, or is dispersed an aqueous solution of the drug. The resultantsolution or dispersion is then sprayed via a nozzle into a dryingchamber of a spray drier to volatilize the organic solvent in the finedroplets in a very short time. This method can be conducted in a mannersimilar to those described in, for example, JP 01-155942A (1989), JP05-194200A (1993), JP 05-70363A (1993), JP 08-151321A (1996), JP09-221417A (1997), U.S. Pat. No. 5,922,253, “Spray Drying Handbook”(John Wiley & Sons, New York 1984), Partick B. Deasy, “Microcapsulationand Related Drug Processes” (Marcel Dekker, Inc., New York 1984) and theforecited Jain et al. (ibid), and the like.

(4) Solvent Diffusion Method

In this method, a solution of a drug and a water insoluble biocompatibleand biodegradable polymer in a water miscible organic solvent is addedto an aqueous solution of protective colloid, followed by emulsificationwith stirring to yield fine particles. This method can be conducted in amanner similar to those described in, for example, JP 05-58882A (1993),JP 09-110678A (1997) and International Journal of Pharmaceutics, vol.187, pp. 143-152 (1999).

In the aforementioned “In-Water Drying Method”, different preparationprocesses may be employed depending on the type of organic phase thoughthey all can be conducted in a conventional manner. Examples of organicphase include the followings.

-   (a) An organic phase wherein a drug is directly dissolved or    dispersed in a solution of a water-insoluble, biocompatible and    biodegradable polymer. This, when dispersed in an aqueous phase,    gives O/W emulsion (JP 56-19324B (1981), JP 63-91325A (1988), JP    06-32732A (1994), JP 08-151321A (1996), JP 06-32732A (1994), and the    forecited Jain, etc.)-   (b) An organic phase which is W/O emulsion wherein an aqueous    solution of a drug is dispersed in a solution of a water-insoluble,    biocompatible and biodegradable polymer. The W/O emulsion, when    dispersed in an aqueous phase, gives (W/O)/W emulsion (JP 60-100516A    (1985),; JP 62-201816A (1987), JP 09-221417A (1997), and the    forecited Jain, etc.)-   (c) An organic phase which is O/O emulsion, which uses two or more    water-insoluble, biocompatible and biodegradable polymers, wherein a    drug is dissolved or dispersed in a polymer solution that is    dispersed in the other(s). The O/O emulsion, when dispersed in an    aqueous phase, gives (O/O)/W emulsion (JP 06-211648A (1994)).

By using any of the organic phases above, the emulsification can beachieved by a conventional method, for example, the intermittent shakingmethod, the method using a mixer such as a propeller shaker or a turbineshaker, the colloidal mill method, the homogenizer method and theultrasonication method.

Examples of organic solvent usable in these methods include halogenatedhydrocarbons (methylene chloride, chloroform, carbon tetrachloride,chloroethane, dichloroethane, trichloroethane, etc.), aliphatic esters(ethyl acetate, butyl acetate, etc.), aromatic hydrocarbons (benzene,etc.), aliphatic hydrocarbons (n-hexane, n-pentane, cyclohexane, etc.),ketones (methylethyl ketone, etc.), ethers (diethyl ether, diisopropylether, methyl isobutyl ether, etc.)

In preparation of emulsion above, an emulsifier may be added to anaqueous phase to stabilize emulsion, which emulsifier includes, forexample, anionic surfactants (sodium oleate, sodium stearate, sodiumlauryl sulfate, etc.), nonionic surfactants {polyoxyethylene sorbitanfatty acid ester [Tween80, Tween.60 (Nikko Chemicals, Co., Ltd.)],polyethylene castor oil derivatives [HCO-60, HCO-50 (Nikko Chemicals,Co., Ltd.)], polyvinylpyrrolidone, polyvinyl alcohol, carboxymethylcellulose, methyl cellulose, lecithin, gelatin, etc.

Further, when one or more other ingredients are incorporated in additionto PDE4 inhibitor, the former can be preferably added to the organicphase at the time of preparation of O/W emulsion. To obtain amicrosphere preparation with an elevated concentration of medicinalingredient, it is necessary to prepare an organic phase containing anactive ingredient at high concentration. For this purpose, anosmoregulatory agent may be included in an aqueous phase to prevent theoutflow of an active ingredient into an aqueous phase (JP 2608245).

The O/W emulsion obtained in the above-mentioned manner is thensubjected to in-water-drying to remove organic solvent present inemulsion to give microspheres.

Organic solvent can be removed from emulsion in a conventional mannersuch as heating, placing under reduced pressure, blowing air, or thelike, and for example, a method where a solvent is distilled off in anopen system (JP 56-19324B (1981), JP 63-91325A (1988), JP 08-151321A(1996), JP 06-211648A (1994)) or in a closed system (JP 09-221418A(1997)) can be employed. In addition, a method where a solvent isextracted and removed by means of a large quantity of outside waterphase (JP-2582186) can also be used.

Further, the following methods can be appropriately used depending onthe PDE4 inhibitor.

A method wherein a solution containing a drug, a biodegradable polymerand a water-miscible good solvent (Solvent A: acetone, tetrahydrofuran,etc.) for the said polymer is first added to a homogeneous mixedsolution comprising a poor solvent (Solvent B: water, ethanol, etc.) forthe said polymer, which is miscible with solvent A, and a poor solvent(Solvent C: glycerin, etc.) for the said polymer, which is immisciblewith solvent A. The mixture, upon emulsification, gives emulsion whereinthe polymer solution constitutes the dispersed-phase and the homogeneousmixed solution constitutes the continuous-phase. The solvent A is thenremoved from the dispersed phase (WO/01/80835)

A method for preparing microspheres from emulsion by in-water-dryingmethod, in which emulsion an organic phase containing an organic solventwith a boiling point lower than water (methylene chloride, ethylacetate, etc.) and a water insoluble polymer is emulsified in an aqueousphase, comprising (1) employing a device equipped with a gas separationmembrane (permeable evaporation membrane, porous membrane, etc.), (2)providing emulsion to be subjected to the in-water-drying to one side ofthe gas separating membrane, and (3) distilling off the organic solventin emulsion to the other side of the gas separating membrane(WO/01/83594)

Furthermore, the organic solvent remaining in microspheres can beremoved by heating microspheres in an aqueous phase at temperaturehigher than the boiling point of the organic solvent (JP 2000-239152A)or heating the microspheres to dry after coating with an additive ofhigh melting point (JP 09-221417A (1997)).

The resultant microspheres are recovered by centrifugation, filtrationor sieving, washed to remove substances attached on the surface such asadditives in the water-phase, and subjected to lyophilization optionallyafter combining with an aggregation inhibitor to prevent theagglomeration of microspheres, for example, sugar, sugar alcohol orinorganic salt, preferably lactose, mannitol or sorbitol. It ispreferred to use a sieve to obtain microspheres of an intended particlesize, and it is more preferred to use a sieve allowing particles of, forexample, 150 μm or below to pass so as to improve the syringeabilitywhen the microsphere preparation is used as injectable solution.

For preparing microspheres by “Phase Separation Method”, amphiphilicsolvents such as acetone, acetonitrile, tetrahydrofuran and dioxane inaddition to the organic solvents used in the “In-water Drying Method”above can be used. A PDE4 inhibitor and optionally one or moreadditional ingredients, or a solution thereof, are dissolved ordispersed in an organic solution of water insoluble polymer in any oneof these organic solvents to form an organic phase. The organic phaseis, added gradually to a solvent (disperse medium) immiscible with theorganic solvent above, for example, silicon oil, liquid paraffin, sesameoil, soybean oil, corn oil, cotton seed oil, coconuts oil, linseed oil,with stirring to form O/O emulsion. If desired, a surfactant may beadded to the disperse medium. The water insoluble polymer can besolidified by cooling the emulsion or evaporating the solvent in theorganic phase by heating. Alternatively, a hardening agent such ashexane, cyclohexane, methyl ethyl ketone, octamethyl-cyclotetrasiloxaneor the like can be added gently to emulsion with stirring, or viceversa, to separate out the water insoluble polymer from emulsion therebyforming microspheres.

The resultant microspheres are recovered by centrifugation, filtrationor sieving, washed with hexane or purified water to remove solvents,additives, etc. attached on its surface, and optionally subjected toair-drying, vacuum-drying, or lyophilization. Alternatively, it can belyophilized after adding an aggregation inhibitor in a manner similar tothat used in the above-mentioned in-water-drying method.

Examples of internal organic phase in the phase separation methodinclude the following embodiments.

-   (a) An organic phase wherein a drug is directly dissolved or    dispersed in a solution of a water-insoluble, biocompatible and    biodegradable polymer.-   (b) An organic phase which is W/o emulsion wherein an aqueous    solution of a drug is dispersed in a solution of a water-insoluble,    biocompatible and biodegradable polymer.-   (c) An organic phase which is O/O emulsion, which uses two or more    water-insoluble, biocompatible and biodegradable polymers, wherein a    drug or a solution thereof is dissolved or dispersed in a polymer    solution that is dispersed in the other(s).

Further, the preparation of microspheres by “Spray Drying Method” isconducted using the same organic solvent as the above-mentioned phaseseparation method. To an organic solvent is dissolved a water insolublebiocompatible and biodegradable polymer, and a PDE4 inhibitor andoptionally one or more additional ingredients, or a solution thereof,are dissolved or dispersed in the solution, and sprayed via a nozzleinto a drying chamber of a spray drier to volatilize the organic solventto form microspheres.

For the present invention, any commercially available spray dryers, forexample, such as Pulvis Mini Spray GS31 (YAMATO Scientific Co., Ltd.),Mini Spray Dryer (Shibata Scientific Technology Ltd.), can be used.

The resultant microspheres are then worked-up in a manner similar tothat used in the in-water drying method to yield the desired microspherepreparation.

Examples of water-miscible organic solvents usable in the “SolventDiffusion Method”, include acetone, methanol, ethanol or a mixturethereof, which may further contain a volatile solvent (methylenechloride, chloroform) in which a drug can dissolve, if necessary.Examples of colloid protective agent include polyvinyl alcohol.

When the microsphere preparation of the present composition forregenerative treatment of cartilage disease, which comprises a PDE4inhibitor as an active ingredient, is administered to a vicinity ofaffected region (especially, in the articular cartilage), it can bepreferably applied locally, more preferably, into articular cartilage asinjection or implant.

An injectable preparation of microspheres can be prepared bydispersing/suspending microspheres obtained by the present invention ata concentration of 0.0001-1000 mg/ml, preferably 0.0005-800 mg/ml, morepreferably 0.001-500 mg/ml into an aqueous solution containing adispersant.

Examples of dispersant include nonionic surfactants such aspolyoxyethylene sorbitan fatty acid ester (Tween80, Tween60, NikkoChemicals Co., Ltd.), polyethylene castor oil (HCO-60, HCO-50, NikkoChemicals Co., Ltd.), cellulose-derived dispersants such ascarboxymethyl cellulose sodium, sodium alginate, dextran, sodiumhyaluronate, and the like. These dispersants can serve to improve thedispersibility of microspheres and stabilize the elution of an activeingredient. A dispersant can generally be added to a composition at aconcentration of 0.01-2% by weight preferably 0.05-1% by weight.

The injectable preparation above may optionally contain a preservative(methylparaben, propylparaben, benzyl alcohol, chlorobutanol, sorbicacid, boric acid, amino acid, polyethylene glycol, etc.), an isotonizingagent (sodium chloride, glycerin, sorbitol, glucose, mannitol, etc.), apH modifier (sodium hydroxide, potassium hydroxide, hydrochloric acid,phosphoric acid, citric acid, oxalic acid, carbonic acid, acetic acid,arginine, lysine, etc.), a buffer (sodium hydrogen phosphate, potassiumhydrogen phosphate, etc.) or the like.

If necessary, a steroid antiinflammatory analgesic or non-steroidalantiinflammatory analgesic may be dissolved or dispersed in theinjectable preparation. Examples of steroidal antiinflammatory analgesicinclude dexamethasone, triamcinolone, triamcinolone acetonide, halopredone, paramethasone, hydrocortisone, prednisolone, methylprednisolone,betamethasone, and the like. Examples of non-steroidal antiinflammatoryanalgesic include ibuprofen, ketoprofen, indomethacin, naproxen,piroxicam, and the like.

In addition to the above-mentioned suspension, the microsphere injectioncontaining PDE4 inhibitor can be in the form of a kit for preparing aninjectable preparation at the time of use, which kit comprises a solidpreparation of an aggregation inhibitor and microspheres, a dispersantand injectable distilled water.

The solid preparation used in a kit can be prepared by suspendingmicrospheres in an aqueous solution containing an aggregation inhibitor,and subjecting the suspension to lyophilization, vacuum drying, spraydrying, and/or the like. The lyophilization is especially preferred.

When preparing a solid preparation, a dispersant can be added to anaqueous solution containing aggregation inhibitor (mannitol, sorbitol,lactose, glucose, xylitol, maltose, galactose, sucrose, etc.) in orderto improve the re-dispersibility into injectable distilled water,thereby yielding a solid preparation of good dispersibility. Ifnecessary, it can be formulated into a kit for preparing an injectablepreparation, in which a steroidal antiinflammatory analgesic and/or anon-steroidal antiinflammatory analgesic as well as a dispersant arecombined.

The present composition for regenerative treatment of cartilage disease,which comprises a PDE4 inhibitor as an active ingredient, can be used intreatment of various warm blood mammals such as human, a domestic animal(a horse, a bull, a sheep, a pig), a pet (a dog, a cat), and the like.The composition for regenerative treatment of cartilage disease can beused in regenerative treatment of various cartilage diseases such asosteoarthrosis, chondrodystrophy, degenerative discopathy, meniscusinjury or the like, and be preferably used in regenerative treatment ofosteoarthrosis.

EXAMPLES

The following Experimental Examples, Examples and Test Examples areprovided to further illustrate the present invention. Throughout thefollowing examples, a compound with a given number is the same compoundindicated by the same number in the list above which shows specificexamples of preferred compounds with chemical structure.

Experimental Example 1 Increase of Matrix Production of ArticularChondrocytes

Test Compounds Compound(1) (10⁻⁵M or 10⁻⁴M); Compound(2) (10⁻⁶M or10⁻⁵M); Compound(9) (10⁻⁶M or 10⁻⁵M); Compound(11) (10⁻⁶M); Compound(21)(10⁻⁶M or 10⁻⁵M); Compound(27) (10⁻⁶M or 10⁻⁵M); Compound(44) (10⁻⁵M or10⁻⁴M);(Isolation and Maintenance of Articular Chondrocytes)

Four NZW line rabbits (Kitayama Labes., Co Ltd.; male; 4-week-old) weresacrificed with bleeding under ether anesthesia and femur knee jointswere collected aseptically. The collected knee joints were stored inphosphate buffer (pH 7.2) containing 0.2% glucose and only the corticallayer of knee joint was scrapped with a surgical knife into a 50 ml tubecontaining phosphate buffer (pH 7.2) containing 0.2% glucose. Thecollected knee joint cortical layer was cut into as small sections aspossible on a dish with a razor and shaken at 37° C. for 15 minutes inphosphate buffer containing 0.2% glucose, supplied with10×trypsin−ethylenediamine tetraacetic acid (EDTA)·4 Na salt(GIBCO; Cat.No. 15400-054) (50 ml, with 100 mg of trypsin and 40 mg of EDTA·4Na; pH7.2). After shaking, the sample was centrifuged (1,400 rpm) to collectprecipitates, and the precipitates were washed twice with 40 ml ofphosphate buffer containing 0.2% glucose. The washed precipitates werecombined with 40 ml of serum-free α-minimum essential medium (MEM:GIBCO; Cat. No. 12571-063) containing 60 mg of collagenase for celldiffusion (Wako Pure Chemical Industries, Ltd., 034-10533) andtransferred to a 100 ml beaker containing a sterilized stirrer bar.Under stirring with the stirrer bar, the collagenase digestion wascarried on for about 1 hour in a CO₂ incubator at 37° C. Cartilagefragments were removed from the collagenase-treated cells using a 40 μMCell Strainer ([FALCON; Cat. No.2340]). To the residual treated cellswas added 10 ml of α-MEM medium containing 10% fetal calf serum (FCS),and centrifuged (1,400 rpm). The precipitates were washed with 10%FCS-αMEM medium twice, suspended in appropriate volume of the samemedium and the resultant suspension was seeded into collagen type II(Wako Pure Chemical Industries, Ltd., 033-13901)-coated plates (48 well)at 20,000 cells/well. On the next day, the medium was replaced with 10%FCS-αMEM medium.

(Increase of Matrix Production)

When cells reached to confluent after the medium exchange procedureabove, the medium for test group was replaced with testcompound-containing medium (including 0.1% dimethylsulfoxide as avehicle). As a medium to which a test compound is added, 10% FCS-αMEMmedium containing 0.2 mM ascorbic acid was used. The day on which testcompound-containing medium was added for the first time was defined as“day 1”. The medium exchange with the same medium was again conducted atday 3 and the cultivation continued until day 5. As to the controlgroup, the medium was exchanged at the same time using the same mediumas the test group except that it is free of test compound (containingvehicle only), and the cultivation was carried out in the same manner.After completion of cultivation, the supernatant was removed from theculture medium. Cells were fixed by addition of 0.25 ml of neutralbuffer containing 4% paraformaldehyde and incubation for 2 hours. Cellswere washed three times with 1 ml of phosphate buffer (pH 7.2) and thenstained for 4 hours with 0.1% Alcian blue 8GX (Sigma; A3157) dissolvedin 0.1 M hydrochloric acid, which Alcian blue selectively stainscartilage matrix proteoglycan. After staining, the cells were washed 3times with 1 ml of phosphate buffer (pH 7.2). Alcian blue which hadstained cartilage matrix was dissolved with 0.25 ml of aqueous 6 Mguanidine hydrochloride solution and a portion of the solution was usedto determine the absorbance at 620 nm. The amounts of Alcian blue usedfor staining was calculated from the absorbance, which in turn was usedfor the estimation of the amount of matrix (proteoglycan). The resultsare shown in Table 1.

TABLE 1 Concentration Proteoglycan Test Compound (M) Production (%)Vehicle — 100 Compound(1) 1 × 10⁻⁵ 133 1 × 10⁻⁴ 144 Compound(2) 1 × 10⁻⁶125 1 × 10⁻⁵ 128 Compound(9) 1 × 10⁻⁶ 128 1 × 10⁻⁵ 138 Compound(11) 1 ×10⁻⁶ 112 Compound(21) 1 × 10⁻⁶ 115 1 × 10⁻⁵ 120 Compound(27) 1 × 10⁻⁶129 1 × 10⁻⁵ 131 Compound(44) 1 × 10⁻⁵ 125 1 × 10⁻⁴ 116

As shown in Table 1 above, it was demonstrated that all the testcompounds (Compounds (1), (2), (9), (11), (21), (27) and (44)) havingPDE4 inhibitory activity exert matrix production promoting activity.

Experimental Example 2 Fractionation of cAMP-hydrolyzing PDE Expressedin Articular Cartilage

Four NZW line rabbits (Kitayama Labes., Co Ltd.; male; 4-week-old) weresacrificed with bleeding under ether anesthesia and femur knee jointswere collected aseptically. The collected knee joints were stored inphosphate buffer (pH 7.2) containing 0.2% glucose and only the corticallayer of knee joint portion was scrapped with a knife into a 50 ml tubecontaining phosphate buffer (pH 7.2) containing 0.2% glucose. Thecollected knee joint cortical layer was cut into as small sections aspossible on a dish with a razor, washed with ice-cold phosphate bufferand homogenized with a homogenizer (Polytron: Kinematica A.G.) inhomogenization buffer (20 mM Tris-HCl, pH 7.4, 2 mM magnesium acetate,0.3 mM calcium chloride, 1 mM dithiothreitol, 40 μM leupeptin, 1.3 mMbenzamidine, 0.2 mM phenylmethylsulfonyl fluoride and 1 mM sodiumazide). The resultant homogenate was centrifuged (100,000×g, 60 minutes)to separate supernatant.

The supernatant was subjected to Mono Q Sepharose High Performancecolumn (Amersham Pharmacia Biotech) previously equilibrated with anelution buffer (20 mM Tris-HCl, pH 7.4, 1 mM calcium chloride, 1 mMdithiothreitol, 2 μM leupeptin, 5 mM benzamidine). After washing thecolumn with 20 ml of elution buffer, proteins were eluted into 1 mlfractions by sodium chloride gradient under ice-cooling. Each fractionwas subjected to the determination of cAMP hydrolyzing activity (PDEactivity) on as a substrate.

The determination of PDE activity was performed by a radio-labelednucleic acid assay. That is, the reaction was initiated by adding from10 to 30 μl of elution fraction to 500 μl of assay buffer [50 mMTris-HCl, pH 8.0, 5 mM magnesium chloride, 4 mM 2-mercaptoethanol, 0.33mg/ml fetal bovine albumin (fatty acid-free, Sigma), 1 mM ethyleneglycolbis(β-aminoethylether)-N,N,N′,N′-tetra-acetic acid] containing 1 μM ofunlabeled cAMP and 22 μM of [³H] -cAMP (Amersham Pharmacia Biotech). Forthe control group, no drug was added while for the test group,Compound(44) was added at a final concentration of 1×10⁻⁵ M. Afterincubation at 37° C. for 30 minutes, the reaction was quenched byboiling for 1.5 minutes. Then, 100 μl of 1 mg/ml Crtalus atrox snakevenom was added and incubated at 37° C. for 30 minutes. After additionof 500 μl of methanol, the reaction mixture was subjected to Dowexcolumn (1×8-400). To each eluate was added a liquid scintillationcocktail and the radio activity was measured. The results are shown inFIG. 1.

FIG. 1 shows that there are four peaks showing strong cAMP hydrolyzingactivity. These four peaks fulfill the features of: (1) havinghydrolytic activity selective to cAMP; (2) said cAMP hydrolyzingactivity being free from the influence of cGMP; and (3) said activitybeing strongly inhibited by Compound(44) that is a selective PDE4inhibitor, and hence were considered to be PDE4-related peaks. It wasreported that PDE4 includes four subtypes, that is, PDE4A, PDE4B, PDE4Cand PDE4D (Saldou et. al., Cellular Signaling, Vol. 10, 427-440, 1998),and these four peaks were assumed to be such subtypes or splicingvariants originated therefrom.

Experimental Example 3 Expression of A Gene Encoding Cartilage MatrixProtein

Total RNA was extracted with ISOGEN (Nippon Gene Co., Ltd.) from rabbitknee articular chondrocytes cultured for 4 days according to the samemanner as Experimental Example 1 in the presence of Compound(1) at afinal concentration of 1×10⁻⁴ M or 1×10⁻⁵ M, and 15 μg of the total RNAwas dissolved in 4.5 μl of a sterilized water. This solution wascombined with 2 μl of 5×MOPS buffer, 3.5 μl of formaldehyde and 10 μl offormamide, and denatured at 90° C. for 15 minutes. The mixture was thenelectrophoresed on 1% agarose gel in the presence of formaldehyde. Aftercompletion of electrophoresis, RNA was transferred to a nylon membrane(Amersham Pharmacia Biotech) overnight by capillary method. The RNA wasfixed to the nylon membrane by UV crosslinking and subjected toprehybridization at 60° C. for 2 hours in 50 ml of hybridizationsolution (6×SSC, 5×Denhart's solution, 0.5% SDS, 100 μg/mlheat-denatured salmon sperm DNA, free of 50% formamide).

Then, DNA probes of mouse type II collagen gene and human aggrecan(typical protein consisting proteoglycan) gene were respectivelyradio-labeled with α[³²P]dCTP using Random-Prime Labeling Kit Ver.2(Amersham Pharmacia Biotech). Each probe (1×10⁸ dpm) and 5 ml ofhybridization solution were added to a prehybridized nylon membrane andsealed, and allowed to hybridize at 60° C. overnight. The nylon membranewas washed with a solution containing 0.2×SSC and 0.2% sodium dodecylsulfate at 60° C. for 40 minutes three times. The nylon membrane wassubjected to an autoradiography and exposed to X-ray film using LAS-1000(Fuji Photo Film Co., Ltd.) A relative amount of each RNA was measuredusing Image Gause (Fuji Photo Film Co., Ltd.) and corrected with 28S RNA(internal RNA: internal control). The gene expression rate in test groupwas calculated by assuming that in control group as 100%. The resultsare shown in Table 2.

TABLE 2 Expression of Type II Expression of collagen gene (%) aggrecangene (%) Control 100 100 Compound(1) 10⁻⁵M 278 2693 Compound(1) 10⁻⁴M406 4262

As shown in Table 2 above, in the test group wherein a PDE4 inhibitor(Compound(1)) was added, the expression of type II collagen and aggrecangenes increased in a dose-dependent manner, which indicated that a PDE4inhibitor affects articular chondrocytes so as to increase geneexpression of type II collagen and aggrecan, which are major componentsof articular cartilage matrix, and thereby promoting the cartilagematrix production.

Experimental Example 4 Increase of Knee Articular Cartilage Matrix inOld Rabbit

(Acclimation)

Old JW line rabbits (Kitayama Labes., Co Ltd.; male; 37-week-old) werehoused at room temperature (23±2° C.) and 50±20% humidity. During thehousing period, the rabbits were free to access commercially availablefood (Oriental Bio; CE-2).

(Increase of Articular Cartilage Matrix Production)

The rabbits were anesthetized by an intravenous injection of Nembutal(Dainabot Co., Ltd.; 50 mg/kg/ml) into an ear vein. The left kneearticular cartilage portion was shaved and sterilized with 70% aqueousethanol. For the test groups, 250 μl of the test compound-containingmicrosphere dispersion (drug content: 2.5 mg) prepared in Example 2-(3)was injected intra-articularly with a 18 gage needle (TerumoCorporation). For the control group, 250 μl of test compound-freemicrosphere dispersion prepared in Control Example 1-(2) was injectedintra-articularly. At 14 days after administration, rabbits weresacrificed with bleeding under Nembutal anesthesia. The knee joints wereisolated, fixed in neutral buffer containing 10% formaldehyde anddecalcified with aqueous 0.5 M EDTA-4Na solution to obtain sections. Thesections were stained with 0.1 M hydrochloric acid containing 0.10%Alcian blue 8GX (Sigma; A3157) which selectively stains cartilage matrix(proteoglycan) and the stainability between the test and control groupswas compared microscopically. As a result, the thickness of the matrixlayer (proteoglycan), which was stained with Alcian blue, was more thanthree times in test groups compared with the control groups.

Experimental Example 5 Regeneration of Knee Articular Cartilage Matrixin Old Rabbit

(Acclimation)

Old JW line rabbits (Kitayama Labes., Co Ltd.; male; 37-week-old) werehoused at room temperature (23±2° C.) and 50±20% humidity in a rabbitcage (C type: W370×D520 ×H330). During the housing period, the rabbitswere free to access commercially available food (Oriental Bio; CE-2).

(Regeneration of Articular Cartilage)

The rabbits were anesthetized by an intravenous injection of Nembutal(Abbott Laboratories; 50 mg/kg/ml) into an ear vein. The left kneearticular cartilage portion was shaved and sterilized with 70% aqueousethanol. The median ligament of left knee was incised to expose thefemur head and meniscus, and the bleeding from neighbor tissue wasstopped with sterilized cotton. A hole (2 mm diameter and 3 mm depth)was bored in the hollow at the middle of femoral head (un-loadedportion) with a drill (TOYO Associates LTD.: Mr. Meister). The hole waswashed with sterilized saline to remove bone scraps etc. generatedduring boring. The articular capsule and median ligament were suturedwith silk thread and hemostasis and disinfection were conducted withsterilized cotton. Nine days later, for the test groups, 250 μl of thetest compound-containing microsphere dispersion (drug content: 2.5 mg)prepared in Example 2-(3) was injected intra-articularly with a 18 gageneedle (Terumo Corporation). For the control group, 250 μl of testcompound-free microsphere dispersion prepared in Control Example 1-(2)was injected intra-articularly. Rabbits were sacrificed 14 days afteradministration with bleeding under Nembutal anesthesia. The knee jointswere isolated, fixed in neutral buffer containing 10% formaldehyde anddecalcified with aqueous 0.5 M EDTA-4Na solution to obtain sections. Thedegree of regeneration at the hole was observed microscopically. Theresults are shown in FIG. 2. As shown in FIG. 2, an advancedregeneration of hole was confirmed clearly in the test groups comparedwith the control group.

Experimental Example 6 Regenerative Healing Effects on Papain-InducedGonarthrosis

(Acclimation)

Japanese White rabbits (Kitayama Labes., Co Ltd.; male; 13-week-old)were housed for 8 days at room temperature (23±2° C.) and 50±20%humidity. During the housing period, the rabbits were fed withcommercially available food (RC4, Oriental Yeast, Co., Ltd.) at the rateof about 140 g/day.

(Regenerative Healing Effect)

The rabbits were anesthetized by an intravenous injection of Nembutal(Abbott Laboratories; Lot. 791102) into an ear vein. The both kneeportions were shaved and sterilized with 70% aqueous ethanol. Therabbits received injections of 0.5 ml of aqueous saline solutioncontaining 0.8% papain (Merck EC 3.4.22.2 lot 587644 019) twice intoboth knee joints at an interval of five days. One week after the secondinjection, for the test group, the microsphere dispersion prepared inExample 2-(3) (containing 0.2 or 2 mg of Compound(1)) was injectedintra-articularly (left knee; 4-6 rabbits/group). For the control group,compound-free microsphere dispersion prepared in Control Example 1-(2)was injected intra-articularly (right knee; 4-6 rabbits/group) in thesame amount as the dispersion used in test group. Furthermore, for theArtz-treated group, 0.3 ml of 1% aqueous hyaluronic acid sodium saltsolution (Artz, Kaken Pharmaceutical Co., Ltd.) was injectedintra-articularly (left knee; 2 rabbits per group). For the non-Artztreated group, 0.3 ml of saline was injected intra-articularly (rightknee; 2 rabbits per group). To both of the Artz-treated andnon-Artz-treated group, the same intra-articular injection as the abovewas conducted weekly four times in total. Four weeks after the lastinjection, rabbits were sacrificed with bleeding under ether anesthesia,knee joints were isolated and fixed in neutral buffer containing 10%formaldehyde. As a result, it was observed that the papain treatmentcaused an articular cartilage degeneration characterized by theirregularity of articular cartilage surface, decreased hematoxylin andeosin stainability, cartilage matrix fibrosis and disappearance ofarticular chondrocytes. Although a slight inhibition in the decrease ofhematoxylin and eosin stainability was observed in the Artz-treatedgroup, remarkable regenerating effects could not be confirmed. On theother hand, in the test group, wherein Compound(1)-containingmicrosphere was administered, showed great improvement of theabove-mentioned pathological symptoms.

In the control group or non-Artz treated group, recovering effects werenot observed at all.

Experimental Example 7 Fractionation of cAMP Hydrolytic PDE Expressed inHuman Articular Cartilage

From the human knee articular cartilage was isolated on surgery ofosteoarthrosis patients. Cartilage portion was scrapped with a surgeryknife, washed with ice-cold phosphate buffer and stored at −80° C. Thecartilage tissues were milled at −80° C. and then, scattered into pieceswith homogenizer (Kinematica A.G., Polytron) in ice-cold homogenizationbuffer (20 mM Tris-HCl, pH 8.0, 1 mM ethylene glycolbis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid, 1 mM dithiothreitol,10 μg/ml leupeptin, 5 mM benzamidine, 0.2 mM phenylmethylsulfonylfluoride, 1 mM sodium azide and 5 mM mercaptoethanol). The resultanthomogenate was centrifuged (100,000×g; 30 minutes) to separatesupernatant.

The above supernatant was loaded on a Mono Q Sepharose High Performancecolumn (Amersham Pharmacia Biotech) equilibrated with elution buffer (20mM Tris-HCl, pH 8.0, 1 mM ethylene glycolbis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid, 1 mM dithiothreitol,2 μg/ml leupeptin, 5 mM benzamidine). After washing the column with 20ml of elution buffer, proteins were eluted into 1 ml fractions byaqueous sodium chloride solution (concentration gradient: 0 to 1000 mM,70 ml) under ice-cooling. Each fraction was subjected to thedetermination of hydrolytic activity (PDE activity) toward cAMP and cGMPas a substrate.

The determination of PDE activity was performed by a radio-labelednucleic acid assay. That is, the reaction was initiated by adding from10 to 50 μl of elution fraction to 500 μl of assay buffer (50 mMTris-HCl, pH 8.0, 5 mM magnesium chloride, 4 mM 2-mercaptoethanol)containing 1 μM of unlabeled cAMP and 22 nM of [³H]-cAMP (AmershamPharmacia Biotech).

After incubation at 37° C. for 30 minutes, the reaction was quenched byboiling for 1.5 minutes. Then, 100 μl of 1 mg/ml Crtalus atrox snakevenom was added and incubated at 37° C. for 30 minutes. After additionof 500 μl of methanol, the reaction solution was subjected to Dowexcolumn (1×8 -400). To each eluate was added a liquid scintillationcocktail and the radio activity was measured. The results are shown inFIG. 3.

As shown in FIG. 3, it was revealed that, in a sample solution preparedby treating human cartilage derived from osteoarthrosis patient, nofractions having cGMP hydrolyzing activity exist, while fractions withpotent cAMP hydrolyzing activity do.

The inhibitory activity of PDE4 inhibitor on fraction Nos. 28-30containing potent cAMP hydrolyzing activity was measured according tothe above-mentioned radio-labeled nucleic acid assay. The test compoundsare Compound(1), Compound(2), Compound(11), Compound(44) and Compound(27).

As a result of experiments, it was confirmed that Compound(1) andCompound(2), in particular Compound(2) inhibits the hydrolytic activityof the fraction strongly. Further, the IC₅₀, of Compound(1) and Compound(2) for the fraction(s), which was measured according to a methoddescribed in Journal of Medicinal Chemistry, vol. 42, 1088-1099 (1999),was consistent with the IC₅₀ of PDE4 inhibition activity described inthe same literature. The results are shown in FIG. 4.

Experimental Example 8 Increase of intracellular cAMP in Human ArticularChondrocytes

(Isolation of Articular Chondrocytes)

Human articular cartilage (articular cartilage of degenerative malumcoxae patient) was soaked in phosphate buffer (pH 7.2) and only thecortical layer of the joint portion was scrapped with a knife into a 50ml tube containing same buffer. The collected knee joint cortical layerwas cut into as small sections as possible on a dish with a razor andtransferred to a centrifuge tube.

To the centrifuge tube, phosphate buffer (pH 7.2) containing 1 mg/ml ofhyaluronidase (SIGMA: Cat. No. H-3506) was added and shaken at 37° C.for 15 minutes. The precipitates were separated by centrifugation (2,000rpm, 5 minutes) and added to Hank's balanced salt solution (GIBCO; Cat.No. 15050-065) containing 0.25% trypsin and shaken at 37° C. for 30minutes.

After separation of precipitates by centrifugation (2,000 rpm, 5minutes), α-minimum essential medium (GIBCO; Cat. No. 12571-063)containing 0.25% collagenase for cell diffusion (Wako Pure ChemicalIndustries, Ltd., 034-10533) and 10% fetal calf serum (GIBCO; Cat. No.10099-141) were added to the precipitates and shaken at 37° C.overnight.

Cartilage fragments were removed using a 40 μm Cell Strainer (FALCON;Cat. No. 2340), and α-minimum essential medium containing 10% fetal calfserum was added to the collagenase-treated cells and centrifuged (1,400rpm, 10 minutes).

The precipitates were washed three times with α-minimum essential mediumcontaining 10% fetal calf serum and suspended in the same medium to anappropriate volume and seeded into 48-well plates (50,000 cells/well).On the next day, the medium was replaced with α-minimum essential mediumcontaining 10% fetal calf serum. The α-minimum essential medium usedcontained antibiotics (100 U/ml penicillin G and 100 μg/ml streptomycinsulfate) and an antifungal (0.25 μg/ml amphotericin B) (GIBCO; Cat. No.15240-062).

(Increase of Intracellular CAMP Concentration)

When cells reached to confluent after the medium exchange procedureabove, the medium for test group was replaced with α-minimum essentialmedium containing 10% fetal calf serum (including 0.1%dimethylsulfoxide) supplied with 1 μM PGE₂ (SIGMA, Cat. No. P-0409) and10⁻⁶ M or 10⁻⁵ M test compound. As to the control group, the medium wasreplaced with a-minimum essential medium (including 0.1%dimethylsulfoxide) containing 10% fetal calf serum-supplied with 1 μMPGE₂ (SIGMA, Cat. No. P-0409).

After 30 minutes cultivation, medium was discarded. The resultingculture was washed with phosphate buffer (pH 7.2) and treated with 50%ethanol for 30 minutes. Ethanol was collected and the ethanol extractwas evaporated to dryness. The residue was dissolved in an assay bufferattached with CAMP EIA system (Amersham Pharmacia Biotech; Cat. No.RPN225) and CAMP concentration was measured with said system. Theresults are shown in Table 3.

TABLE 3 Test PGE₂ cAMP Production Compound Concentration (1 μM)(picomol/well) Vehicle 0 + 0.4 Compound(2) 1 × 10⁻⁶ + 13.8 1 × 10⁻⁵ +23.2 Compound(11) 1 × 10⁻⁵ + 10.6

Experimental Example 9 Matrix Production of Rabbit ArticularChondrocytes in the Presence of IL-1

Four NZW line rabbits (Kitayama Labes., Co Ltd.; male; 4-week-old) weresacrificed with bleeding under ether anesthesia and knee joints of femurside were collected aseptically. Only the cortical layer was scrappedwith a surgical knife in phosphate buffer (pH 7.2) containing 0.2%glucose and then placed into a 50 ml tube containing phosphate bufferedsaline containing 0.2% glucose. The collected knee joint cortical layerwas cut into as small sections as possible on a dish with a razor,combined with 50 ml of phosphate buffer (100 mg trypsin, 40 mg EDTA·4Na; pH 7.2) containing 0.2% glucose, supplied with10×trypsin-ethylenediamine tetraacetic acid tetra sodium salt (EDTA-4Na:GIBCO; Cat. No.15400-054) and shaken at 37° C. for 15 minutes.

After shaking, the precipitates were collected by centrifugation (1,400rpm) and washed twice with 40 ml of phosphate buffer (pH 7.2) with 0.2%glucose.

To the washed precipitates was added 40 ml of α-minimum essential medium(MEM: GIBCO; Cat. No. 12571-063) containing 60 mg of collagenase forcell diffusion (Wako Pure Chemical Industries, Ltd., 034-10533) andantibiotics (200 U/ml penicillin G and 200 μg/ml streptomycin sulfate)(GIBCO; Cat. No. 15140-122), and the mixture was transferred to a 100 mlbeaker containing a sterilized stirrer bar.

The incubation was conducted in a CO₂ incubator at 37° C. for 30 minutesunder stirring with a stirrer bar. Deoxyribonuclease I (Takara ShuzoCo., LTD.; Cat. No. 2210A) was then added at a concentration of 70 U/ml.The cultivation was conducted under the same condition for another 30minutes. The supernatant of the culture was collected in another vesseland the remaining cartilage slips were then cultured again for about 30minutes in freshly prepared a-minimum essential medium containing 60 mgof collagenase and 70 U/ml deoxyribonuclease I.

To the previously collected culture supernatant and the last culture,from which cartilage slips had been removed using a 40 μm Cell Strainer(FALCON; Cat. No.2340), 10 ml of α-minimum essential medium (MEM: GIBCO;Cat. No. 12571-063) containing 10% fetal calf serum (GIBCO Cat. No.10099-141) and antibiotics (200 U/ml penicillin G and 200 μg/mlstreptomycin sulfate) (GIBCO; Cat. No. 15140-122) was added andcentrifuged (1,400 rpm, 10 minutes).

The precipitates were washed twice with α-minimum essential mediumcontaining 10% fetal calf serum and antibiotics, suspended with anappropriate volume of the same medium, and seeded into 48-well plates(20,000 cells/well). On the next day, the medium was replaced with thesame medium.

(Increase of Matrix Production)

Following the above medium exchange procedures, when cells reached toconfluent, the medium of test group was replaced with a medium(including 0.1% dimethylsulfoxide) supplied with 1 ng/ml recombinanthuman IL-1β (PEPRO TECH; Cat. No. 200-01B) and a test compound. As themedium, α-minimum essential medium containing fetal bovine serum andantibiotics (supra), and also 0.2 mM ascorbic acid was used. On theother hand, in the control group, the medium was replaced with the samemedium as the test group except that a test compound was not added.

The day when IL-1β containing medium was added was defined as “day 1”and the cultivation-was continued until day 3.

After the cultivation, the supernatant was removed from the culture andcells were fixed by addition of 0.25 ml of 10% neutral buffered formalinsolution (Wako Pure Chemical Industries, Ltd., Cat. No. 060-01667).

The fixed cells were washed three times with 1 ml of phosphate buffer(pH 7.2) and stained for 4 hours with 0.1% Alcian blue 8GX (Sigma;A3157) ) dissolved in 0.1 M hydrochloric acid, which Alcian blueselectively stains matrix (proteoglycan).

After staining, the cells were washed 3 times with 1 ml of phosphatebuffer (pH 7.2). Alcian blue which had stained cartilage matrix wasdissolved with 0.25. ml of 6 M aqueous guanidine hydrochloride solutionand a portion of which was used to determine the absorbance at 620 nm.The amount of Alcian blue used for staining was calculated from theabsorbance, which in turn was used for the, estimation of the amount ofmatrix (proteoglycan). The relative percentage was calculated byassuming the proteoglycan production in control group, wherein a testcompound was not added, to be 100%. The results are shown in Table 4.

TABLE 4 Concentration IL-1β Proteoglycan Test Compound (M) (1 ng/ml)Production (%) Vehicle 0 + 100 Compound(2) 1 × 10⁻⁶ + 143 Compound No.53 1 × 10⁻⁶ + 211 Compound No. 56 1 × 10⁻⁶ + 168 Compound No. 52 1 ×10⁻⁶ + 209 Compound No. 57 1 × 10⁻⁶ + 169 Compound(11) 1 × 10⁻⁶ + 121

As shown in the table 4 above, in the presence of IL-1, the testcompound having PDE4 inhibitory activity increased the matrixproduction. It, is thought that IL-1 plays a important role in cartilagematrix degradation, because IL-1 is expressed in synovial fluid andcartilage cells of osteoarthrdsis patients, and induces the productionand synthesis of matrix metalloproteinase (MMP), which is matrix (suchas cartilage matrix, proteoglycan) catabolic enzyme (The Journal ofPharmacology and Experimental Therapeutics, vol 277, pp. 1672-1675,1966; Journal of Biochemistry, vol 123, pp. 431-439, 1998; Arthritis &Rheumatism, vol 44, pp. 585-594, 2001). Therefore, the results describedabove suggested that a PDE4 inhibitor, which is an active ingredient ofthe present invention, has inhibitory activity against IL-1-relatedcartilage matrix degradation.

Experimental Example 10 Regenerative Healing Effects on GonarthrosisInduced by Partial Excision of Medial Meniscus/Abscission of BilateralCollateral Ligaments

(Acclimation)

Japanese White rabbits (male; 10-week-old; 7 rabbits/group) were housedfor 16 days at room temperature (23±2° C.) and 55±15% humidity. Duringthe housing period, the rabbits were free to access commerciallyavailable food (Oriental Bio Service; LRC4).

(Healing of Gonarthrosis)

Under ether anesthesia, the right knee joint of each rabbit was excisedand ½ portion of medial meniscus was isolated with double-edged smallstraight scissors. The bilateral collateral ligaments were also cut.After the operation, muscle and epidermal tissues were sutured andsterilized. Two weeks from the operation, under ether, anesthesia, eachrabbit of the test group (7 rabbits/group) was administeredintra-articularly the drug-containing microsphere prepared in Example7-(1), which contains 1 μg of Compound (2). The rabbit of the controlgroup (7 rabbits/group) received the same amount of drug-freemicrosphere prepared in Control Example 2. Six weeks after theoperation, the above-mentioned drug-containing or -free microsphere wasagain administered. Ten weeks after the operation, under etheranesthesia, rabbits were sacrificed by laparotomy with bleeding and thetibial proximal end was isolated. The isolated tibial proximal end wastreated with India ink and then soaked into 10% neutral bufferedformalin solution for fixation.

Experimental Results

After wiping off the excess India Ink, the overhead surface image of theformalin-fixed tibial proximal end was imported into an analyzer, ImageAnalyzer (IMAGING RESEARCH, MICD imaging analyzer), with astereomicroscope (OLYMPUS OPTICAL Co., Ltd. model SZX12-2111). Thisanalyzer was used to measure the area of medial portion where India inkremains (the degenerated area). The gross medial area was also measuredand the percentage (%) (the degenerated area rate) of the degeneratedmedial area in the gross medial area was calculated. The results areshown in Table 5.

TABLE 5 Drug Drug amount Degenerated Area Rate (%) Control 0 μg 18.86 ±2.03 Compound(2) 1 μg 10.16 ± 1.35

Example 1

(1) To 0.1 g of Compound(1) and 1.9 g of lactic acid-glycolic acidcopolymer (lactic acid: glycolic acid=50:50; average molecular weight20,000; PLGA5020: Wako Pure Chemical Industries, Ltd.). was added 4.0 gof methylene chloride, and the mixture was shaken for 30 minutesthoroughly to form an oil phase (O).

(2) The oil phase was added to 8 ml of 0.5% aqueous solution ofpolyvinyl alcohol (POVAL PVA-220C: Kuraray Co., Ltd.) and emulsified at25° C. for 5 minutes with homogenizer (Polytron, Kinematica A.G.) toform (O/W) emulsion, wherein the oil phase is dispersed in the waterphase.

(3) The emulsion was added to 1000 ml of distilled water, stirred at 400rpm with Three-one motor (Shinto Scientific Co., Ltd.) and subjected toin-water drying method at 25° C. for 3 hours to remove methylenechloride.

(4) The resultant microsphere suspension was filtered through 150 μmfilter to remove aggregates and filtered under reduced pressure through20 μm filter to remove the water phase. The resultant microsphere wascombined with a little amount of distilled water and lyophilized to give1.6 g of microsphere.

Ten mg of the resultant microsphere was dissolved in 3 ml ofacetonitrile. The solution was combined with 7 ml of 0.5 M aqueoussodium chloride solution, stirred with a mixer (Touch mixer MT-51:YAMATO Scientific Co., Ltd.) and then centrifuged at 2000 rpm for 5minutes to separate supernatant. A portion of supernatant was loaded onFL-HPLC (column; Hypersil 5-ODS, diameter: 4 mm, length: 300 mm, GLSciences, Inc., excitation wavelength: 315 nm, fluorescence wavelength:465 nm) and the drug concentration in the supernatant was determined bycomparing with a standard curve prepared separately with a drugsolution. On the basis of the resultant concentration and the volume ofsupernatant, the drug content in microsphere was estimated as 4.21%.

An adequate amount of resulting microsphere was dispersed in a dilutesolution of polyoxyethylene sorbitan fatty-acid ester (Tween 80: NikkoChemicals Co., Ltd.) The particle distribution was measured with aparticle size analyzer SALD-1100 (Shimadzu Corporation), and the averageparticle size was calculated. The average particle size was 57 μm.

(5) The microsphere obtained in (4) above was added to physiologicalsaline (dispersion medium) containing 0.5% carboxymethyl cellulosesodium (Nichirin Chemical Industries) and 0.1% polyoxyethylene sorbitanfatty acid ester (Tween 80: Nikko Chemicals Co., Ltd.) at final drugconcentration of 2.5 mg/ml, and the mixture was stirred with a mixer(Touch mixer MT-51: YAMATO Scientific Co., Ltd.) thoroughly to yieldmicrosphere dispersion.

Example 2

(1) Microsphere (1.6 g) was prepared in a manner similar to thatdescribed in Example 1-(1) to (4) except that a mixture of 0.57 g oflactic acid-glycolic acid copolymer (lactic acid:glycolic acid=50:50;average molecular weight 20,000; PLGA5020: Wako Pure ChemicalIndustries, Ltd.) and 1.33 g of lactic acid polymer (average molecularweight 20,000; PLA0020: Wako Pure Chemical Industries, Ltd.) was used.

The drug content and the average particle size of microsphere weremeasured in a manner similar to that described in Example 1-(4) andproved to be 3.70% and 47.7 μm, respectively.

(2) The microsphere obtained in (1) above was treated in a mannersimilar to that described in Example 1-(5) to give microspheredispersion (drug rate: 2.5 mg/ml).

(3) The microsphere obtained in (1) above was treated in a mannersimilar to that described in Example 1-(5) to give microspheredispersion (drug rate: 10.0 mg/ml).

Example 3

(1) Microsphere (1.5 g) was prepared in a manner similar to thatdescribed in Example 1-(1) to (4) except that lactic acid polymer(average molecular weight 20,000; PLA0020: Wako Pure ChemicalIndustries, Ltd.) was used.

The drug content and the average particle size of microsphere weremeasured in a manner similar to that described in Example 1-(4) andproved to be 3.73% and 52.2 μm, respectively.

(2) The microsphere obtained in (1) above was treated in a mannersimilar to that described in Example 1-(5) to give microspheredispersion (drug rate: 2.5 mg/ml).

Example 4

(1) To 0.2 g of Compound(1) and 0.3 g of lactic acid polymer (averagemolecular weight 20,000; PLA0020: Wako Pure Chemical Industries, Ltd.)was added 1.0 g of methylene chloride, and the mixture was shaken with amixer (Touch mixer MT-51: YAMATO Scientific Co., Ltd.) thoroughly toform an oil phase (O).

(2) The oil phase was added to 4 ml of 0.25% aqueous solution of methylcellulose (METOLOSE: Shin-Etsu Chemical Co., Ltd.) and emulsified at 25°C. for 5 minutes with homogenizer (Polytron, Kinematica A.G.) to form(O/W) emulsion, wherein the oil phase is dispersed in the water phase.

(3) The emulsion was added to 400 ml of distilled water, stirred at 400rpm with Three-one motor (Shinto Scientific Co., Ltd.) and subjected toin-water drying method at 25° C. for 3 hours to remove methylenechloride.

(4) The resultant microsphere suspension was filtered through 150 μmfilter to remove aggregates and filtered under reduced pressure through20 μm filter to remove water phase. The resultant microsphere wascombined with a little amount of distilled water and lyophilized to givemicrosphere. The drug content and the average particle size ofmicrosphere were measured in a manner similar to that described inExample 1-(4) and proved to be 39.6% and 33.4 μm, respectively.

Example 5

(1) To 0.05 g of Compound(1) and 0.45 g of lactic acid-glycolic acidcopolymer (lactic acid:glycolic acid=50:50; average molecular weight20,000; R202H: Boehringer Ingelheim Co., Ltd.) was added 1.0 g ofmethylene chloride, and the mixture was shaken with a mixer (Touch mixerMT-51: YAMATO Scientific Co., Ltd.) thoroughly to form an oil phase (O).

(2) The oil phase was added to 40 ml of 0.5% aqueous solution ofpolyvinyl alcohol (GOHSENOL EG-40: The Nippon Synthetic ChemicalIndustry Co., Ltd.) and emulsified at 25° C. for 4 minutes withhomogenizer (Polytron, Kinematica A.G.) to form (O/W) emulsion, whereinthe oil phase is dispersed in the water phase.

(3) Emulsion was poured into a cylindrical airtight container (insidediameter: 110 mm; volume 1,000 ml) containing 400 ml of purified water,and methylene chloride 5 was removed from the container by stirring at25° C. and 400 rpm using 4-bladed propeller (diameter: 50 mm, propellerR type: HEIDON) equipped with Three-one motor (BL-600; HEIDON) whilesupplying nitrogen gas into hollow fibers of cylinder-type hollow fibermembrane module made of silicone rubber (NAGAYANAGI Co., Ltd.) insertedin the container (gas flow rate is 2 L/minute). This procedure wasconducted for 1 hour.

The cylindrical hollow fiber membrane module made of silicone rubberused in this procedure is cylinder type NAGASEP M60-1800 of thefollowing specification.

Cylinder diameter: 100 mm Cylinder length: 120 mm × 120 mm Membranethickness of hollow fiber membrane: 60 μm Inside diameter of hollowfiber membrane: 200 μm Outside diameter of hollow fiber membrane: 320 μmNumber of hollow fiber: 1800 Effective membrane area of hollow fiber0.15 m² membrane:

(4) The resulting microsphere suspension was filtered through 150 μmfilter to remove aggregates and filtered under reduced pressure through20 μm filter to remove water phase. The resultant microsphere wascombined with a little amount of distilled water and lyophilized to give0.26 g of microsphere. The drug content and the average particle size ofmicrosphere were measured in a manner similar to that described inExample 1-(4) and proved to be 3.07% and 71.7 μm, respectively.

Example 6

(1) To 0.05 g of Compound(2) and 0.45 g of lactic acid-glycolic acidcopolymer (lactic acid:glycolic acid=50:50; average molecular weight20,000; RG502H: Boehringer Ingelheim Co., Ltd.), 2.5 g of methylenechloride was added and shaken with a mixer (Touch mixer MT-51: YAMATOScientific Co., Ltd.) thoroughly to form an oil phase (O).

(2) The oil phase was added to 3 ml of 0.5% aqueous solution ofpolyvinyl alcohol (POVAL PVA-220C: Kuraray Co., Ltd.) and emulsified at22° C. for 5 minutes with homogenizer (Polytron: Kinematica A.G.) toform (OW) emulsion, wherein the oil phase was dispersed in the waterphase.

(3) The above procedures (1) and (2) were repeated five times. Theresultant emulsions (from 5 trials) were combined, added to 1000 ml ofdistilled water, and stirred at 400 rpm with Three-one motor (ShintoScientific Co., Ltd.) to remove methylene chloride by conductingin-water-drying at 25° C. for 1.5 hours, at 40° C. for 1 hour and at 25°C. for 0.5 hours.

(4) The resultant microsphere suspension was filtered through 150 μmfilter to remove aggregates and filtered under reduced pressure through20 μm filter to remove water phase. The resultant microsphere wascombined with a little amount of distilled water and lyophilized to give2.3 g of microsphere.

Ten mg of the resultant microsphere was dissolved in 3 ml ofacetonitrile. The solution was combined with 6 ml of aqueous 0.5 Msodium chloride solution, stirred with a mixer (Touch mixer MT-51:YAMATO Scientific Co., Ltd.) and then centrifuged at 2000 rpm for 5minutes to separate supernatant. A portion of supernatant was loaded onUV-HPLC. (column; Hypersil 5-ODS, diameter: 4 mm, length: 300 mm, GLSciences, Inc., detection wavelength: 240 nm) and the drug concentrationin the supernatant was determined by comparing with a standard curveprepared separately with a drug solution. On the basis of the resultantconcentration and the volume of supernatant, the drug content inmicrosphere was estimated. Further, the average particle size wasmeasured in a manner similar to that described in Example 1-(4). As aresult, the drug content was 9.9% and the average particle size was 26.4μm.

(5) The microsphere obtained in (4) above was treated in a mannersimilar to that described in Example 1-(5) to give microspheredispersion (drug rate: 0.1 mg/ml).

Example 7

(1) Microsphere (2.2 g) was prepared in a manner similar to thatdescribed in Example 6-(1) to (4) except that lactic acid-glycolic acidcopolymer (lactic acid:glycolic acid=75:25; average molecular weight20,000; PLGA7520: Wako Pure Chemical Industries, Ltd.) was used and that2.0 g of methylene chloride was added.

The drug content and the average particle size of microsphere weremeasured in a manner similar to that described in Example 6-(4) andproved to be 10.1% and 27.0 μm, respectively.

(2) The microsphere obtained in (1) above was treated in a mannersimilar to that described in Example 6-(5) to give microspheredispersion (drug rate: 0.1 mg/ml).

Control Example 1 Control of Example 2

(1) To 0.6 g of lactic acid-glycolic acid copolymer (lacticacid:glycolic acid=50:50; average molecular weight 20,000; PLGA5020:Wako Pure Chemical Industries, Ltd.) and 1.4 g of lactic acid polymer(average molecular weight 20,000) was added 4.0 g of methylene chloride,and the mixture was shaken for 30 minutes thoroughly to form an oilphase (O). In accordance with the procedures described in Example 1-(1)to (4), 1.7 g of microsphere free of drug was obtained.

(2) The microsphere obtained in (1) above was treated in a sameprocedures described in Example 1-(5) to prepare microsphere dispersion,wherein the dispersed microsphere concentration in the dispersion is thesame as that of Example 2-(3).

Control Example 2 Control of Example 7

To 0.45 g of lactic acid-glycolic acid copolymer (lactic acid:glycolicacid=75:25; average molecular weight 20,000; PLGA7520: Wako PureChemical Industries, Ltd.) was added 2.0 g of methylene chloride, andthe mixture was shaken with a mixer (Touch mixer MT-51: YAMATOScientific Co., Ltd.) thoroughly to form an oil phase. In accordancewith the procedures described in Example 6-(2) to (4), 2.2 g ofmicrosphere free of drug was obtained.

Test Example 1

To 10 mg of microsphere in a test tube was added 10 ml of phosphatebuffer (pH 7.4) containing 0.05% Tween 80, and stirred with a rotatingcultivator at 25 rpm in an air-temperature-controlled cabinet at 37° C.When a defined period of time passed from the initiation of stirring,test tube was centrifuged (2000 rpm, 5 min) and of stirring, test tubewas centrifuged (2000 rpm, 5 min) and 9 ml of supernatant was sampledand loaded on FL-HPLC. (column; Hypersil 5-ODS, diameter: 4 mm, length:300 mm, GL Sciences, Inc., excitation wavelength: 315 nm, fluorescencewavelength: 465 nm) and the drug content was determined by comparingwith a standard curve prepared separately with a drug solution. On thebasis of the result and the sampling volume, the elution amount of drugwas estimated.

Further, the estimation of elution amount of drug was repeated regularlyby adding 9 ml of phosphate buffer (pH 7.4) to the test tube aftersampling, and conducting the same procedures under the same conditions,which comprises stirring, sampling, and estimating.

After the final sampling, the remaining eluate was removed from the testtube and the drug content in the residual microsphere was determinedaccording to the method described in Example 1-(4).

The above procedures were carried out on the microspheres obtained inExamples 1 to 3. The results are shown in FIG. 5.

The elution rate was calculated based on the assumption that the sum ofdrug eluted from and remained in the microsphere being 100%.

Test Example 2

Male SD rats (7-weeks-old, 3 rats/group, Japan SLC) were conditioned fora week by housing at room temperature (23±2° C.) under 12 hourslight-dark cycle while feeding with food and water ad libitum. Each ratthen received rapid-injection of Compound(1) (1 mg/ml) dissolved inphysiological saline containing 10% polyethylene glycol 400 ((Wako PureChemical Industries, Ltd.) from femoral vein at 0.5 ml/animal (totaldrug dosage: 0.5 mg/rat).

After drug administration, under ether anesthesia blood samples werecollected at regular time intervals from jugular vein with a syringecontaining heparin and centrifuged to obtain plasma samples. To 0.1 mlof plasma were added 0.2 ml of internal standard solution and 1 Mdibasic potassium phosphate and then 7.0 ml of chloroform. The mixturewas shaken for 10 minutes and centrifuged for 5 minutes to separate 5 mlof organic phase. The resultant organic phase was evaporated to drynessat 40° C. under nitrogen atmosphere, re-dissolved in 0.5 ml of mobilephase and then loaded on FL-HPLC. (column; Hypersil 5-ODS, diameter: 4mm, length: 300 mm, GL Sciences, Inc., excitation wavelength: 315 nm,fluorescence wavelength: 465 nm) to determine the plasma concentration.The results are shown in FIG. 6.

Test Example 3

Male SD rats (7-weeks-old, 5 rats/group, Japan SLC) were conditioned fora week by housing at room temperature (23±2° C.) under 12 hourslight-dark cycle while feeding with food and water ad libitum. Each ratthen received subcutaneously microsphere dispersion obtained in Examples1-(5), 2-(2) or 3-(2) from back at 2 ml per rat (total drug dosage: 5mg/rat). After drug administration, under ether anesthesia, bloodsamples were collected at regular time intervals from jugular vein witha syringe containing heparin and centrifuged to obtain plasma samples.The concentration of the compound in plasma was determined in a mannersimilar to that described in Test Example 2. As a result of formulatingPDE4 inhibitor into microsphere, the maximum plasma concentration ofPDE4 inhibitor could be reduced to 1/25 to 1/100, even when comparedwith that achieved by intravenous injection of saline containing only atenth amount of PDE4 inhibitor (Test Example 2). The results are shownin FIG. 7.

Test Example 4

Male SD rats (7-weeks-old, 5 rat per group, Japan SLC) were conditionedfor a week by housing at room temperature (23±2° C.) under 12 hourslight-dark cycle while feeding with food and water ad libitum. Each ratthen received subcutaneously microsphere dispersion obtained in Example2-(2) from back at 2 ml per rat (total drug dosage: 5 mg/rat).

At days 3, 7, 10, 14, 21 and 35 after drug administration, microsphereswere collected from the sites of administration. To the collectedmicrospheres, 5 ml of acetonitrile containing internal control substancewas added and dissolved with homogenizer (Polytron: Kinematica A.G.)After centrifugation at 3,000 rpm, 5 minutes, 3 ml of supernatant wascollected, combined with 7 ml of 0.5 M aqueous sodium chloride solution,stirred with a mixer (Touch mixer MT-51: YAMATO Scientific Co., Ltd.)and then centrifuged at 2,000 rpm for 5 minutes to separate supernatant.A portion of supernatant was filtrated through KC. prep-omni 13(Katayama Chemistry Inc.) and loaded on FL-HPLC. (column; Hypersil5-ODS, diameter: 4 mm, length: 300 mm, GL Sciences, Inc., excitationwavelength: 315 nm, fluorescence wavelength: 465 nm). The drugconcentration was determined by comparing with a standard curve preparedseparately with a drug solution. On the basis of the resultantconcentration and the volume of supernatant, the residual rate of a drugremaining in microsphere was calculated. The results are shown in FIG.8.

Test Example 5

Male SD rats (7-weeks-old, Japan SLC) were conditioned for a week byhousing at room temperature (23±2° C.) under 12 hours light-dark cyclewhile feeding with food and water ad libitum. Each rat then receivedsubcutaneously Compound(2)-containing microsphere dispersions obtainedin Examples 6-(5) and 7-(2) at 1 ml per rat (total drug dosage: 0.1mg/rat) from back.

Microspheres were collected at regular time intervals from theadministration site. To the collected microspheres, 10 ml ofacetonitrile was added and dissolved with homogenizer (Polytron:Kinematica A.G.) After centrifugation at 3,000 rpm for 5 minutes, 3 mlof supernatant was collected, combined with 6 ml of 0.5 M aqueous sodiumchloride, stirred with a mixer (Touch mixer MT-51: YAMATO ScientificCo., Ltd.) and then centrifuged at 2000 rpm for 5 minutes to separatesupernatant. A portion of supernatant was filtrated through KC.prep-omni 13 (Katayama Chemistry Inc.) and loaded on UV-HPLC. (column;Hypersil 5-ODS, diameter: 4 mm, length: 300 mm, GL Sciences, Inc.,detection wavelength: 240 nm). The drug concentration was determined bycomparing with a standard curve prepared separately with a drugsolution. On the basis of the resultant concentration and the volume ofsupernatant, the residual rate of a drug remaining in microsphere wascalculated. The results are shown in FIG. 9.

INDUSTRIAL APPLICABILITY

Although only conservative treatment has been considered to be availableas drug treatment of cartilage disease, the present composition forregenerative treatment of cartilage disease, which comprises a PDE4inhibitor as an active ingredient, especially when administered locallyto the affected cartilage region, makes it possible to regenerate thecartilage without producing side effects due to systemic action of PDE4inhibitor, whereby exerts regenerative therapeutic effects on cartilagediseases especially osteoarthrosis. Still higher effect can be achievedby formulating a composition containing a PDE4 inhibitor and abiocompatible and biodegradable polymer into a depot preparation,especially into an injectable microsphere preparation, administering thesame locally to an affected cartilage region thereby allowing efficacyto last.

1. A method for regenerative treatment of cartilage disease in apatient, which comprises local administration to an affected region of acomposition comprising a PDE (phosphodiesterase) 4 inhibitor as anactive ingredient and a biocompatible and biodegradable polymer forgradually releasing the PDE4 inhibitor at the affected region.
 2. Themethod according to claim 1, which is for regenerative treatment ofosteoarthrosis.
 3. The method according to claim 1, wherein thebiocompatible and biodegradable polymer is water-insoluble.
 4. Themethod according to claim 3, wherein the composition comprises amicrosphere preparation.
 5. The method according to 4, wherein theparticle size of a microsphere is 0.1-150 μm.
 6. The method according toclaim 4, wherein the composition is an injectable microspherepreparation comprising the microspheres at a concentration of0.0001-1,000 mg/ml in an aqueous solution comprising a dispersant. 7.The method according to claim 6, wherein the injectable microspherepreparation comprises the dispersant at a concentration of 0.01-2% byweight.
 8. The method according to claim 6 or 7, wherein the dispersantis one or more selected from the group consisting of polyoxyethylenesorbitan fatty acid ester, polyethylene castor oil, carboxymethylcellulose sodium, sodium alginate, dextran and sodium hyaluronate. 9.The method according to claim 4 or 5, wherein the PDE4 inhibitor contentis 0.0001-80% by weight of the composition.
 10. The method according toclaim 9, wherein the PDE4 inhibitor is2,3-bis(hydroxymethyl)-6,7-diethoxy-1-[1-(2metoxyethyl)-2-oxo-4-pyridyl]inaphthaleneor 2,3-bis(hydroxymethyl)-6,7-diethoxy-1-[2-(4-(3-pyridyl)-1(2H)-phthaladinon-2-yl)-4-pyridyl]naphthalene or a pharmaceuticallyacceptable salt thereof.
 11. The method according to claim 3, whereinthe water-insoluble biocompatible and biodegradable polymer is a hydroxyfatty acid polyester.
 12. The method according to claim 3, wherein thewater-insoluble biocompatible and biodegradable polymer is one or morepolymers selected from the group consisting of poly lactic acid, lacticacid-glycolic acid copolymer and 2-hydroxybutyric acid-glycolic acidcopolymer.
 13. The method according to claim 11 or 12, wherein thewater-insoluble biocompatible and biodegradable polymer has an averagemolecular weight of 2,000-800,000.
 14. The method according to claim 1,wherein the PDE4 inhibitor is a selective PDE4 inhibitor.
 15. The methodaccording to claim 1, wherein IC₅₀ of the PDE4 inhibitor is less than100 nM.
 16. The method according to claim 1, wherein the PDE4 inhibitoris a compound having a partial structure having PDE4 inhibitory activityas follows: (A) naphthalene or an analogous chemical structure thereof;or (B) 3-cyclopentyloxy-4-methoxyphenyl or an analogous chemicalstructure thereof.
 17. The method according to claim 1, wherein the PDE4inhibitor is a compound, which has PDE4 inhibitory activity, having apartial structure of naphthalene or isoquinoline skeleton, or apharmaceutically acceptable salt thereof.
 18. The method according toclaim 1, where the PDE4 inhibitor is a compound of one of the formula(I)-(III) or a pharmaceutically acceptable salt thereof:

wherein R¹ and R² are the same or different and each a hydrogen atom, ahydroxyl group, a cyclo-lower alkyloxy group, or an optionallysubstituted lower alkoxy group, or bind together at the ends to form alower alkylenedioxy group; R³ is an optionally substituted 6-memberednitrogen-containing heterocyclic group; and —OR⁴ and —OR⁵ are the sameor different and each an optionally protected hydroxyl group;

wherein R¹′ and R²′ are the same or different and each a hydrogen atomor an optionally protected hydroxyl group; either of R³′ or R⁴′ is anoptionally protected hydroxy-substituted methyl group and the other is ahydrogen atom, a lower alkyl group or an optionally protectedhydroxy-substituted methyl group; and R⁵′ and R⁶′ are the same ordifferent and each a hydrogen atom, an optionally substituted loweralkyl group, an optionally substituted phenyl group or an optionallyprotected aminio group, or bind together at the ends and form inassociation with the adjacent nitrogen atom an optionally substitutedheterocyclic group;

wherein A is a group selected from those shown by the formulas:

wherein R¹″ and R²″ are the same or different and each a hydrogen atomor an optionally protected hydroxyl group; R³¹ is an optionallyprotected hydroxymethyl group; R³² is a hydrogen atom, a lower alkylgroup or an optionally protected hydroxymethyl group; R³³ is anoptionally substituted lower alkyl group; R⁴¹ is an optionally protectedhydroxymethyl group; R⁴² is an optionally protected hydroxymethyl group;the dotted line represents the presence or absence of a double bond; andR⁵″ and R⁶″ are the same or different and each a hydrogen atom or anoptionally protected amino group, or bind together at the ends and formin association with the adjacent nitrogen atom an optionally substitutedheterocyclic group.
 19. The method according to claim 1, wherein thePDE4 inhibitor is one selected from the group consisting of Compounds(1)-(57) or a pharmaceutically acceptable salt thereof:


20. A method for preparing a lyophilized product for use in aninjectable preparation, comprising suspending a composition comprising aPDE (phosphodiesterase) 4 inhibitor as an active ingredient, awater-insoluble biocompatible and biodegradable polymer, and amicrosphere preparation, wherein the composition is configured forgradually releasing the PDE4 inhibitor at an affected region in thepatient for regenerative treatment of cartilage disease, in an aqueoussolution comprising an aggregation inhibitor; and lyophilizing theresultant suspension.